This invention relates to a method for producing sodium hyaluronate by a fermentation method and, in more detail, to a method for producing and accumulating sodium hyaluronate by culturing a mutant strain derived from Streptococcus equi ATCC 9527 and partially free from auxotrophy.
Heretofore, sodium hyaluronate was extracted from rooster's comb, vitreous bodies of eyes of cattle or umbilical cord. However, the preparation of sodium hyaluronate by extraction has the drawback of highly complicated isolation and purification.
To overcome this drawback, there is proposed in Japanese Unexamined Patent Publications Nos. 56692/1983 and 63294/1986 a method consisting of culturing a microorganism having the ability of producing sodium hyaluronate and directly collecting sodium hyaluronate from the culture liquid. However, this method has the drawback that the amount of production is unstable from lot to lot and it is difficult to practice the method industrially. Furthermore, a solution containing sodium hyaluronate obtained by a fermentation method contains high molecular compounds and metals as impurities. Such impurities need to be separated and removed. To this end, various proposals have been made in the art, such as the method of adsorption by ion exchange resins, activated charcoal, diatomaceous earth or magnesium silicate, the method of treatment with quaternary ammonium salts or proteases, the method of extraction or precipitation by solvents such as ethanol, acetone, butanol or phenol, or the method of treatment with membrane filters.
These methods, however, are complicated in purification and are unsatisfactory for preparing sodium hyaluronate of high purity.